Color changing polymer films for detecting chemical and biological targets

ABSTRACT

A sensor system, and a method of detecting a target analyte, comprises a chemically functionalized block copolymer, and a target analyte. The block copolymer exhibits a color change in the visible spectrum upon exposure to the target analyte.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/484,533, filed May 31, 2012, which application is based on U.S.Patent Application Ser. No. 61/492,728, filed Jun. 2, 2011, entitled“Color Changing Polymer for Chemical/Biological Threat and PathogenDetection,” which applications are incorporated herein by reference intheir entireties and to which priority is claimed.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported by the US Army ECBC ILIR Program as provided forby the terms of W911SR08C0031. This work was also supported by theNational Science Foundation Grant No. CBET0947771. The US government hascertain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to a sensor system that exhibits a visiblechange upon exposure to a target, and in particular, a chemicallyfunctionalized block copolymer film that exhibits a color change in thevisible light spectrum upon exposure to a target analyte.

BACKGROUND OF THE INVENTION

Conventional sensor systems have been developed from various materialsthat change color upon exposure to a target molecule. Such sensorsystems are generally based on photonic crystals that have been modifiedto recognize the target molecule. The photonic crystal contains periodicnanostructures with differing indices of refraction that interact withvisible light. The material recognizes or binds to specific chemicaltargets. Recognition of the target alters the spacing of the periodicnanostructure, thereby changing the way it interacts with visible light.

The advantage of such systems lies in producing a discernable change incolor. This concept has been applied in the fabrication of colloidalhydrogel systems, porous silicon systems, as well as through the use oflithography techniques on other substrates. The use of a photoniccrystal polymerized colloidal hydrogel system has been demonstrated (Cuiet al. (2009) Analyst 134(5):875-880; Lee, K., Asher, S. A. (2000)Journal of the American Chemical Society 122(39):9534-9537. Suchmaterial could act as a sensor for glucose but required the complexprocess of synthesizing monodisperse, highly charged polystyreneparticles. Porous silicon (Lee, M., Fauchet, P. M. (2007) Optics Express15 (8), 4530-4535; Li et al. (2003) Science 299(5615): 2045-2047) andnanoprint lithography (Endo et al. (2010) Sensors and Actuators B:Chemical 148(1):269-276) have also been reported as photonic crystalchemical sensing platforms. The observed response in such conventionalsystems, however, is small, non-visible, and thus cannot be measuredwithout the aid of supplementary equipment and further analyticalmeasurements.

Therefore, there is a need for a sensor that can be easily fabricated,and yield an instantaneous, visibly discernable response.

SUMMARY OF THE INVENTION

The present invention is directed to hybrid biotic/abiotic structuresfabricated using chemically functionalized block copolymers, such asPolystyrene-b-poly(2-vinyl pyridine) (PS-b-P2VP) block copolymers. Thepolymer films of the present invention induce a visible color changewhen exposed to aqueous media.

In disclosed embodiments, the P2VP block of the copolymer may befunctionalized with either 2-bromomethylphenylboronic acid orbromoethylamine. In one implementation, the 2-bromomethylphenylboronicacid functionalization allows the polymer films to respond to glucosewith a change in color. For example, when exposed to glucose the colorof the film may be changed from green to orange.

In one implementation, ovalbumin antibodies are attached to filmsfunctionalized with bromoethylamine. These films respond to theovalbumin protein with a color change. This functionalization may befurther modulated to detect foodborne pathogens, such as Escherichiacoli, Listeria, Salmonella, warfare agents such as Ricin, Sarin orSoman, or HMEs such as nitroglycerin or TATP. The polymer coatingundergoes a color change in the visible light spectrum, providingmanufacturers, consumers, and vendors with a facile method foridentifying such analytes.

A sensor system according to one embodiment of the disclosed inventioncomprises a chemically functionalized block copolymer, and a targetanalyte. The block copolymer exhibits a color change in the visiblespectrum when exposed to the target analyte. In one implementation, theblock copolymer has a lamellar morphology or structure.

In one embodiment, the functionalized block copolymer is apolystyrene-b-poly(2-vinyl pyridine) (PS-b-P2VP) block copolymer. In oneimplementation, the P2VP block of the copolymer is functionalized with2-bromomethylphenylboronic acid or bromoethylamine.

In one embodiment, the target analyte is a sugar, such as for example,glucose, fructose, galactose and mannose. In another embodiment, thetarget analyte is a foodborne pathogen, such as for example Escherichiacoli, Listeria, or Salmonella. In another embodiment, the target analyteis a toxin, such as for example, ricin, sarin, or soman. In anotherembodiment, the target analyte is an explosive compound, such as forexample nitroglycerin or triacetone triperoxide. In another embodiment,the target analyte is a component of human sweat.

In one embodiment, an antibody is linked to the functionalized blockcopolymer, wherein the antibody binds to the target analyte. In oneimplementation, the antibody is polyclonal ovalbumin antibody.

In one embodiment the functionalized block copolymer is coupled to atextile or fabric material. In one implementation, the resulting textilematerial exhibits a shift in color change upon exposure to human sweat.

A method of detecting a target analyte according to an embodiment of thepresent invention comprises the steps of: providing a block copolymer;functionalizing the block copolymer; and exposing the functionalizedblock copolymer to a target analyte, whereby the functionalized blockcopolymer exhibits a color change in the visible light spectrum uponexposure to the target analyte.

In one embodiment of the disclosed method, the block copolymer is2-vinyl pyridine. The 2-vinyl pyridine block copolymer may befunctionalized with 2-bromomethylphenylboronic acid or bromoethylamine.

The present invention is also directed to a chemically functionalizedblock copolymer. The functionalized block copolymer exhibits a shift inpeak wavelength in the visible light spectrum upon exposure to a targetmolecule. In one implementation, the shift in peak wavelength is atleast about 40 nm. In one implementation, the shift in peak wavelengthis at least about 200 nm.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing/photographexecuted in color and that copies of this patent with colordrawing(s)/photograph(s) will be provided by the Office upon request andpayment of the necessary fee.

FIG. 1 is a schematic of a BCP film sensor according to an embodiment ofthe present invention.

FIG. 2 is a schematic of BCP film according to the present invention,showing the film in an unbound state (shown at the left) and a boundstate (shown at the right). The spacing (d) and refractive indexcontrast between subsequent layers changes when a chemical/biological(CB) agent selectively binds to capture reagent in one of the layers.

FIG. 3 illustrates the functionalization of the 2-vinyl pyridine blockwith: 2-Bromomethylphenylboronic acid, shown in FIG. 3(a); andbromoethylamine, shown in FIG. 3(b).

FIG. 4 is a graph showing Fourier transform infrared spectroscopy (FTIR)spectrum data of an unmodified annealed polymer film compared to a filmmodified with bromoethylamine/bromomethylphenylboronic acid.

FIG. 5 illustrates schematically PS-b-2VP films quaternized withbromoethylamine and further modified with polyclonal ovalbumin antibodyusing a carbodiimide reaction. When introduced to ovalbumin protein, theprotein binds to the antibody causing a shift in mass and volume in thepolymer film, causing the film to swell and change color (from orange tored).

FIG. 6 shows antibody modified PS-b-2VP film having a light orangecoloration (left plate) before exposure to protein, and a deep redorange coloration (right plate) after exposure to ovalbumin.

FIG. 7 is a graph showing FTIR spectrum data of an unmodified annealedpolymer film compared to a film modified withbromoethylamine/bromomethylphenylboronic acid.

FIG. 8 illustrates schematically the PS-b-2VP film functionalized with2-Bromomethylphenylboronic acid, whereby the boronic acid groupsreversibly bind to each other, cross-linking the film and inhibitingswelling. When exposed to glucose, the boronic acid cross-links arebroken as the boronic acid groups bind to glucose, thereby allowing thefilm to swell.

FIG. 9 shows PS-b-2VP film functionalized with2-Bromomethylphenylboronic acid having a green color (left plate), andan orange color (right plate) after exposure to a 50 mg/ml D-glucosesolution.

FIG. 10 illustrates graphically the shift in the visible spectrum's peakof the films before and after exposure to glucose measured usingUltraviolet-visible (UV-Vis) spectroscopy. FTIR spectrum data of film inwater and in a 50 mg/ml glucose solution is shown in FIG. 10A; FTIRspectrum data of film in water and a 40 mg/ml glucose solution is shownin FIG. 10B.

FIG. 11 illustrates graphically UV-Vis spectrum data of films in waterand various glucose solutions. The film changed from a red color to anorange color upon exposure to 30 mg/ml (Plate A), from red-orange toyellow upon exposure to 20 mg/ml (Plate B), from orange to yellow uponexposure to 10 mg/ml (Plate C), from red to orange upon exposure to 5mg/ml (Plate D), and exhibited minimal color change upon exposure to 1mg/ml (Plate E).

FIG. 12 illustrates transmission electron microscopy (TEM) images ofannealed, unmodified PS-b-P2VP film (shown in Plate A), and of PS-b-P2VPfilm functionalized with 2-bromomethylphenylboronic acid exhibiting alamellar morphology (shown in Plate B). The darker iodine stained P2VPblock appears thicker due to the increased mass of the P2VP block fromthe functionalization as compared to the cross section seen in theunmodified film.

FIG. 13 illustrates graphically the colored BCP films and theircorresponding visible spectra. As shown, the BCP film color can be tunedto blue, green, yellow, orange or transparent (infrared) colorsdepending on the degree of crosslinking. Increasing the crosslinkdensity inhibits swelling, which blue-shifts the color of the polymerfilm. Each BCP film displays a different color and visible spectradepending on the molar ratio of crosslinker to quatenizer.

FIG. 14 illustrates the UV-Visible spectrum of the functionalized filmin both water and glucose solution. When exposed to pure water, the filmswelled due to the positive charge placed on the P2VP block from thefunctionalization (Plate A). Once immersed in a glucose solution, thefunctionalized polymer film swelled further due to the boronic acidgroups binding glucose (Plate B). When bound to glucose, a negativecharge exists on the boron atom of the boronic acid moiety, causing thefilm to further swell and become orange in color (Plate C). In water thefilm swells to reflect a peak wavelength of 510 nm, which corresponds toa color green. In the glucose solution, the film swells and reflects apeak wavelength of 590 nm, which corresponds to an orange color.

FIG. 15 illustrates graphically in graphs A-F the shift in peakwavelength of BCP samples immersed in various concentrations of glucosesolution of: 50 mg/ml (A); 40 mg/ml (B); 30 mg/ml (C); 10 mg/ml (D); 5mg/ml (E); and 1 mg/ml (F). 50 mg/ml and 40 mg/ml solutions caused anincrease, or red-shift, in peak wavelength, indicating that the polymerfilm had swelled. However, a decrease, or blue-shift, was observed inthe BCP film samples exposed to 30 mg/ml, 10 mg/ml, and 5 mg/mlsolutions, indicating that the film collapsed.

FIG. 16 illustrates graphically the shift in peak wavelength ofinitially tuned blue BCP films exposed to four different sugar solutionscontaining glucose, fructose, galactose or mannose at the sameconcentration. The difference of the peak wavelength observed in waterversus that observed in the sugar solution was calculated to obtain theshift in wavelength. As shown, exposure to fructose induced the largestresponse of an approximately 200 nm red-shift in wavelength. Galactoseinduced a 70 nm increase in wavelength, and mannose and glucose inducedsmaller increases of approximately 40 nm each. This indicates that theBCP film can differentiate between fructose, galactose, mannose andglucose for a given concentration.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a polymeric material capable ofspecifically and selectively recognizing a variety of distinct targets,such as foodborne pathogens, chemical and biological warfare agents, andhomemade explosives (HMEs). The disclosed material may be in form of aflexible polymer film or coating, which may be applied to foodpackaging, provided as a litmus test strip, configured as small“stickers” or large coating sheets, or integrated into fabric. Uponselective recognition of a target, for example foodborne pathogens suchas Escherichia coli, Listeria, Salmonella, warfare agents such as Ricin,Sarin or Soman, or HMEs such as nitroglycerin or triacetone triperoxide(TATP), the polymer material undergoes a color change in the visiblelight spectrum providing manufacturers, consumers, and vendors with afacile method for identifying any of these analytes or other selectedtargets.

The visible light spectrum (or visible spectrum or visible light) is theportion of the electromagnetic spectrum that is visible to the humaneye. Generally, a human eye will respond to and perceive wavelengthsfrom about 390 to 750 nm (corresponding to a band in the vicinity of400-790 THz). The visible color change in the visible spectrum of blockcopolymers is triggered by changes in swelling conditions of thematerial. The recognition properties, stability and reusability of knownbinding moieties specific to each analyte create a simple and reliablenanostructured polymer material or coating providing utility in a widevariety of applications.

In order to create a polymeric material that can successfully recognizea desired biological or chemical molecule, a selective recognitionelement is combined with a measurable output signal. According to anembodiment of the present invention, the disclosed materials achievethese needs by utilizing the tunable reflectance of swollenfunctionalized block copolymers (BCPs). Block copolymers include two ormore chemically distinct sequences of monomer repeat units linkedtogether through a covalent bond. Upon evaporation from a solvent, BCPswill microphase separate into solid films, displaying a number ofdifferent morphologies (e.g., hexagonal, cubic, gyroid, lamellar)depending on the relative volume fraction of each block (Bates et al(1990) Annual Review of Physical Chemistry 41:525-557).

BCPs in which both blocks are of equal molecular weight generallyexhibit the lamellar morphology. Self-assembly into a lamellarmorphology is significant in producing a BCP photonic crystal. If thereis enough contrast in refractive index between the two blocks in thelamellar structure, then certain wavelengths of light will be reflectedby the material. This phenomenon is dictated by: λ₁=2(n₁d₁+n₂d₂), whereλ₁ is the reflected wavelength, n_(i) is the refractive index of layer iand d_(i) is the thickness of layer i.

In one embodiment, the diblock copolymerpolystyrene-b-poly(2-vinylpyridine) (PS-b-P2VP), which microphaseseparates into a lamellar periodic stack, was utilized for its use as achemical sensor to detect and respond to glucose with a change in color.The 2-vinyl pyridine (P2VP) block was quaternized with2-(bromomethyl)phenylboronic acid, which placed a positive charge on thepyridine ring of the block. This charge allows the BCP film to swell inaqueous media. The swelling changes the thickness of the block allowingit to interact with wavelengths of visible light. Lamellar PS-b-P2VPfilms quaternized with bromoethane place a positive charge in the P2VPblock, and attach an ethyl group to the nitrogen atom. The quaternizingagent contributes a boronic acid residue, giving the BCP the ability tobind to sugars such as glucose. Binding induced a change in the distancebetween the lamellae, causing a change in the wavelength light reflectedby the polymer, thus allowing the BCP to act as a glucose sensor.

Boronic acids are of interest in chemical sensing due to their abilityto covalently bind to sugar molecules such as glucose (see Bosch et al.(2004) Tetrahedron 60(49): 11175-11190; Chen et al. (2009) Langmuir25(12):6863-6868; Kim et al. (2007) Chemical Communications22:2299-2301). Although sensing glucose has applications in diabeticmedicine, the utilization of glucose sensing using the boronic acidfunctionalized BCP system served as a model system to test the conceptthat block copolymer based photonic crystals can be fabricated to act aschemical sensors for small molecule detection. In the present invention,the polymer film was tested for successful attachment of the boronicacid, retention of the lamellar morphology postfunctionalization, andsensitivity and specificity to simple sugar binding.

A schematic of a BCP film sensor according to an embodiment of thepresent invention is shown in FIG. 1. Initially, the BCP film isfabricated to exhibit a periodic lamellar stack. The P2VP block of theblock copolymer is functionalized with phenylboronic acid placing apositive charge on the pyridine ring, which allows the P2VP block toswell in aqueous media until its thickness is large enough to interactwith visible light, in this case reflecting green light. Thephenylboronic acid can bind sugars and will do so when exposed to aglucose solution.

As shown in FIG. 1, the boronic acid binds to the 1,3 diolfunctionality. This is the kinetically favored binding site on glucoseas well as the 1,2 cis diol (see Bielecki et al. (1999) Journal of theChemical Society: Perkin Transactions 2(3):449-455). This binding eventlowers the pKa of the phenylboronic acid causing it to form thenegatively charged boronate complex. The negative charge triggersadditional swelling of the BCP film, changing its color from green toorange. We have shown that after functionalization, the BCP film canrespond to a glucose solution and shows a selective response whenexposed to different sugars, such as fructose, mannose or galactose.

Thus, an A-B diblock copolymer that microphase separates into a lamellarperiodic stack is utilized. The A block is water-insoluble polystyrene(PS) and does not contain any functional groups on the polymer chainthat can interact with the target (e.g., biological/chemical threatcompound). The B block contains the 2-vinyl pyridine (P2VP)functionality, which can be further chemically modified to serve asionic interaction sites with known threat receptors.

Without modification, the refractive index contrast between subsequent Aand B layers is too low to successfully reflect light in the visiblewavelength. In addition, the spacing between these layers is not largeenough to interact with visible light. Through selective chemicalmodification of the B block, the condition for visible light can beachieved, thus creating lamellar structures with tunable reflectiveproperties. Upon binding an analyte, the B layers swell, increasing thespacing between the subsequent A layers. In addition, the refractiveindex contrast between alternating layers is increased. The ranges ofwavelengths reflected are highly dependent upon the spacing between theA and B layers, and the refractive indices of subsequent layers. Thus,the color of the self-assembled polymer films may be modulated by simplychanging the spacing (d) between subsequent layers, as shown in FIG. 2.This may be achieved by controlling the amount of swelling due to thetarget pathogen (e.g., biological or chemical) binding event.

With continued reference to FIG. 2, films of the block copolymerpolystyrene-b-poly(2-vinyl pyridine) (PS-b-P2VP) may be fabricated tohave a periodic lamellar structure. These solid films may then bechemically functionalized. This functionalization only occurs on thepoly(2-vinyl pyridine) block of the copolymer, placing a positive chargeon the nitrogen atom of the pyridine ring, thereby causing the film toswell in water and change color.

In one implementation, the block copolymer films were functionalizedwith either 2-bromomethylphenylboronic acid or bromoethylamine, as shownin FIG. 3. The choice of either 2-Bromomethylphenylboronic acid orbromoethylamine depends on the chemical or biological target. The2-(bromomethyl)phenylboronic acid functionalization allows the films totarget sugars (e.g., such as glucose), while the bromoethylaminefunctionalization allows for further modification of the films.

For example, to functionalize the P2VP block of the copolymer, asolution of 40 mg of either 2-(bromomethyl)phenylboronic acid orbromoethylamine in 40 mL of acetonitrile is prepared. The blockcopolymer films are then immersed in the solution for 24 hours at 90° C.The 90° C. temperature is a solubility LCST for the PS-b-P2VP inacetonitrile. This temperature prevents the polymer from dissolving inthe solvent, thereby permitting functionalization.

When functionalized with the 2-bromomethylphenylboronic acid, the filmsrespond to the sugar glucose with a change in color. The bromoethylaminefunctionalization places a primary amine along the poly-2-vinyl pyridineblock. The primary amine may then have antibodies for proteins, such asRicin, attached to the polymer film. When exposed to Ricin, the CB agentwill bind to the antibody causing the polymer film to swell and changecolor. In one implementation, polyclonal ovalbumin antibodies areattached to the polymer films, and the films are then exposed to theovalbumin protein. The films respond with a change in color, asdescribed above.

In another implementation, the functionalized block copolymer materialis integrated into or coupled to a fabric material. The resulting fabricmaterial may be integrated into a shirt or other article of clothing,and change color when exposed to a selected target substance. Forexample, a shirt may include a portion (e.g., such as a company logo ordesign) including the polymer material integrated therein, whichexhibits a change in color in the visible spectrum when exposed to thewearer's sweat, for example a target component of sweat.

Having generally described the invention, the same will be more readilyunderstood through reference to the following examples, which areprovided by way of illustration and are not intended to be limiting ofthe present invention unless specified.

EXAMPLES

The use of functionalized photonic block copolymer films for thedetection of glucose was investigated. Polystyrene-b-poly(2-vinylpyridine) (PS-b-P2VP) block copolymers were chemically functionalizedwith 2-(bromomethyl)phenylboronic acid and cast into films that reflecta visible color when exposed to aqueous media. The2-(bromomethyl)phenylboronic acid functionality can reversibly bind toglucose. When exposed to high concentrations of glucose the polymerresponded with a red-shift in color. Low concentration exposure ofglucose caused the polymer films to blue-shift in color. The BCP filmsalso exhibited a selective response to fructose, mannose or galactose,giving a different response depending on which sugar was present. Thecolor of the polymer was tuned to blue, green, yellow or orange byvarying the film's crosslink density. The color change was visuallyobserved without the use of equipment such as a spectrometer.

Fabrication of Photonic BCP Films

The procedure for fabrication of the PS-b-P2VP films was adapted fromKang et al. (2007). The PS-b-P2VP block copolymers were purchased fromPolymer Source (Montreal, Canada). The molecular weight of each block ofthe copolymer was 133,000 g/mol. A 5% weight/volume stock solution ofPS-b-P2VP was prepared in propylene glycol monomethyl ether acetate(PGMEA). The solution was then passed through a 0.2 μm teflon filter anddegassed under vacuum for 10 minutes. The films were prepared byspin-casting 300 μl of the PSb-P2VP solution onto 1″×1″ glass slides at350 rpm for 2 minutes. The glass slides were purchased from Ted Pella(Redding, Calif.), and were functionalized with3-(aminopropyl)triethoxysilane. The spin-cast block copolymer films weresubsequently annealed in chloroform vapor at room temperature (50° C.)for 24 hours to allow them to self-assemble into a lamellar structure.

Functionalization and Tuning of Photonic Properties

The P2VP block of the spincast films was quaternized with2-(bromomethyl)phenylboronic acid. The quaternization reaction places aphenylboronic acid functional group in the P2VP block, allowing thepolymer to bind sugars (e.g., glucose). The quaternization reaction wascarried out by immersing the spincast, annealed block copolymer films ina solution of 40 mg of 2-(bromomethyl)phenylboronic acid in 40 ml ofacetonitrile and allowing it to reflux for 5 h. The quaternized polymerswere then removed from the solution and dried in a 50° C. oven for 1hour.

To tune the optical properties of BCP film a crosslinking agent,1,4-dibromo-2-butanol, was introduced into the acetonitrile solution.Different molar ratios of 2-(bromomethyl)phenylboronic acid to1,4-dibromo-2-butanol were tested at 0.05 mmol as the total monomeramount. The same protocol was used to investigate the effect ofdifferent degrees of crosslinking on the color of the BCP films.

Characterization

Fourier transform infrared spectroscopy (FTIR) was utilized to determinewhether the boronic acid functionality was successfully attached. Thefunctionalized polymer film was removed from the glass slide byimmersion in a 5% v/v aqueous solution of hydrofluoric acid. Thefreestanding polymer film was dried for 24 hours in a 50° C. oven. AnFTIR spectrum was obtained of the functionalized polymer film using aThermo Nicolet FT-Raman, Model 670 (Thermo Fisher Scientific, Waltham,Mass.). To determine whether the lamellar morphology was maintainedafter functionalization, images of the polymer were taken usingtransmission electron microscopy (TEM). The sample was prepared byembedding a section of the functionalized polymer film in Spurr's resinpurchased from Ted Pella (Redding, Calif.). The embedded polymer filmwas then dry microtomed and stained through exposure to solid iodine for1 hour. The images were taken using a JEM-2100F 200 kV (Jeol Ltd.,Tokyo, Japan) transmission electron microscope.

Sugar Detection

Once the films were functionalized with 2-(bromomethyl)-phenylboronicacid, they were exposed to various concentrations of d-glucose toobserve if the films would respond with a color change. Aqueoussolutions of 50, 40, 30, 10, 5 and 1 mg/ml of d-glucose were prepared.The films were soaked in 20 ml of DI water and a coverslip was placed ontop of the polymer film on the glass slide to retain the water in thefilm as it was transferred to a UV-Visible spectrometer. Thewater-swollen control sample was then placed in a Perkin Elmer Lambda25UV-Visible spectrometer and the visible spectrum was measured usingUV-Vis spectroscopy. Each film was then soaked in one of the preparedglucose concentrations and the visible spectrum was again measured andcompared to the film soaked in pure water. To test for selectivitybetween glucose and other sugars, films were prepared and exposed to 50mg/ml solutions of glucose, fructose, galactose and mannose and theirvisible spectra measured.

Antibody Attachment and Protein Detection

Films quaternized with bromoethylamine were further modified byattaching polyclonal ovalbumin antibodies. The attachment was performedusing 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as well asN-Hydroxysuccinimide (NHS). The ovalbumin antibody was placed in(N-morpholino)ethanesulfonic acid buffer solution using a Slide-A-Lyzerat a concentration of 1 mg/ml. 1.1 mg of NHS and 0.5 μl EDC were addedto the solution. The EDC was allowed to bind to the carboxylic acidgroups on the antibody for 15 minutes. A film functionalized withbromoethylamine was then immersed in this solution for 3 hours. Once theantibody attachment reaction was complete, the film was introduced to asolution of 30 mg/ml solution of ovalbumin protein.

Before testing each film's response to either ovalbumin or glucose, FTIRwas utilized to determine if the functionalizations were successful.Once verified, films with the bromoethylamine functionalization werefurther modified with ovalbumin antibodies, and then exposed ovalbuminsolutions, and observed. Films functionalized withbromomethylphenylboronic acid were placed in various glucoseconcentrations, and their responses were measured using UV-Visspectroscopy.

Verification of Bromoethylamine Attachment

To verify that the PS-b-2VP films were functionalized withbromoethylamine, FTIR was utilized to determine functionalization withbromoethylamine (see FIG. 4), as well as antibody attachment.

Detection of Ovalbumin

PS-b-2VP films quaternized with bromoethylamine were further modifiedwith polyclonal ovalbumin antibody using a carbodiimide reaction. Asillustrated schematically in FIG. 5 and shown in FIG. 6, when introducedto ovalbumin the film swelled, changing its color from light orange tored. This concept may be adapted for other proteins and enzymes, such asantibodies specific to Ricin or peroxidases for the detection of the HMETATP.

Verification of Boronic Acid Attachment

To determine if the 2-(bromomethyl)phenylboronic acid was attached tothe glucose sensing polymer, FTIR was utilized (see FIG. 7).

Detection of Glucose

The PS-b-2VP films functionalized with 2-Bromomethylphenylboronic acidwere exposed to a 20 mL 50 mg/mL aqueous solution of D-glucose. Thefilms were initially green in pure deionized water due to thefunctionalization. Once exposed to the glucose solution the filmsinstantly swelled and became orange in color. This color change andproposed mechanism is illustrated schematically in FIG. 8 and shown inFIG. 9.

Films were exposed to various aqueous concentrations of glucose. To showa change in color, the visible spectrum of the films before and afterexposure to glucose was measured using UV-Vis spectroscopy. Thespectrum's peak shifted toward 700 nm or the “red” end of the spectrumafter exposure, as shown in FIGS. 10A and 10B. When exposed toconcentrations of 50 mg/ml and 40 mg/ml glucose solutions, the filmsshifted from green to orange, or green to yellow, respectively. Thisindicates that they became swollen.

Exposure to lower concentrations of glucose solution had a differenteffect. As seen in FIG. 11, for concentration of 30 (FIG. 11A), 20 (FIG.11B), 10 (FIG. 11C) and 5 (FIG. 11D) mg/ml glucose solutions, the filmscollapsed when exposed to the glucose solution. Their color shiftedtowards 400 nm or the “violet” end of the spectrum. Thus, the filmsswelled at higher concentrations and collapsed at lower concentrationsof glucose solutions.

Discussion

In order for maximal diffraction of light toward the observer, thelamellar morphology of the BCP film was oriented parallel to the glasssubstrate. The 3-(aminopropyl)triethoxysilane functionalization on theglass substrate interacts with the P2VP block of the BCP by influencingthe morphology to be parallel to the substrate. The BCP film is thenannealed by exposing it to chloroform vapor, mobilizing the polymerchains and allowing them to form the parallel lamellae.

This annealing process is relatively specific and thus preciseconditions are preferably controlled. Environmental factors such as roomhumidity and evaporation rate of the annealing solvent may cause the BCPfilm to have poor morphology or break the interactions between the BCPand the substrate. It was found that low humidity, approximately 20%,and relatively slowed evaporation rate of the chloroform dramaticallyimproved morphology. After completion of the annealing process, the BCPfilm was chemically functionalized by attaching2-(bromomethyl)phenylboronic acid to the P2VP block. The BCP film wasexposed to acetonitrile at an elevated temperature. However, such harshconditions could potentially damage the BCP film or disrupt its delicatelamellar morphology.

If the lamellar morphology is disrupted by the chemical modification,then the photonic properties of the BCP film will be diminished. TEM wasutilized to determine whether the lamellar morphology was maintainedafter functionalization with the 2-bromomethyl)phenylboronic acid.

Referring to FIG. 12, the electron micrograph of the cross section ofboth an unmodified BCP film and of a BCP film functionalized with2-(bromomethylphenylboronic acid) is presented. The lamellar morphologyneeded for the film's optical properties was maintained afterfunctionalization of the P2VP block. In the unmodified film (FIG. 12A),the layer thickness of the P2VP block and PS block are similar in sizeas the molecular weight of each block is comparable. The iodine-stainedblock is the P2VP.

After chemical functionalization (FIG. 11B), the lamellar morphology wasmaintained. However, there is an apparent increased thickness of thedarker iodine stained P2VP block versus the lighter PS block due to theincreased mass added to the P2VP block from the2-(bromomethyl)phenylboronic acid. Verification that the PS-b-P2VP filmswere functionalized was obtained by analyzing the chemical structurewith FTIR. The pyridine group of the P2VP block substitutes the brominein the 2-(bromomethyl)phenylboronic acid which covalently bonds theboronic acid functionalization to the pyridine. The boronic acidfunctionalization places a positive charge on the nitrogen atom in thepyridine ring of the P2VP block as the pyridine groups are converted topyridinium. This conversion from pyridine to pyridinium can be observedusing FTIR. The FTIR spectrum of a modified and unmodified film wastaken. A peak appeared at 1627 cm⁻¹ in the modified film indicating theconversion of pyridine to pyridinium, or the placement of a formalpositive charge on the nitrogen atom of the pyridine ring.

This functionalization serves two purposes. First, a formal positivecharge is placed on the P2VP block of the BCP film, allowing it to swellin water. This swells the P2VP block to a sufficiently large thickness,which allows it to interact with visible light as dictated by Bragg'slaw. The second purpose of the functionalization is to introduce boronicacid to the BCP film. Boronic acid binds to sugar molecules allowing theBCP film to recognize and respond to simple sugars with a change incolor. This substitution reaction was performed via exposure of the BCPfilm to a solution of 2-(bromomethyl)phenylboronic acid in acetonitrilewhile refluxing for 5 hours. This methodology could be applied for otherchemical functionalizations, each with its own sensing application.

Tuning the Polymer Film Photonic Properties

Control over the optical properties or color of the BCP film allows forthe production of a reliable chemical sensor. To tune the color of thefunctionalized BCP film, varying degrees of crosslinking were introducedinto the P2VP block. The crosslinker used was 1,4-dibromo-2-butanol inconjunction with the boronic acid functionalization. The BCP was exposedto various molar ratios of 1,4-dibromo-2-butanol (crosslinker) to2-(bromomethyl)phenylboronic acid (quaternizer). Increasing ordecreasing the mole fraction of the crosslinker, allows the collapsingor swelling the polymer film in water, which tunes the color of the BCP.

The BCP films may be fabricated to exhibit different colors bycontrolling the swelling of the polymer film through variations incrosslink density. For example, the BCP films may be tuned to blue,green, yellow, orange or infrared colors. Referring to FIG. 13, each ofthe colored BCP films and their corresponding visible spectra areillustrated. As shown, the BCP film's color is intense and readilyrecognizable visually. Thus, the need for extraneous equipment todistinguish one color from another is eliminated. This allows for thefabrication of a relatively simple sensor, which may be easily utilizedby individuals and without the need for specific training to determineif the BCP film has responded to a specific analyte.

Response to Glucose

Boronic acid can bind to 1,2 and 1,3 cis diols, a chemical functionalitycommonly found in sugar molecules. The binding of a sugar to the boronicacid lowers its pKa. The pKa change increases the number of boronic acidresidues that form the boronate complex, which is negatively charged.Such negative charge then causes the BCP film to swell, thereby changingits color. The PS-b-P2VP films functionalized with2-(bromomethyl)phenylboronic acid were exposed to a range ofconcentrations of aqueous d-glucose solutions. The tested polymer filmswere initially green in pure deionized water due to thefunctionalization placing a positive charge on the P2VP block. The greenfilms exposed to the 50 mg/ml glucose solution instantly swelled andbecame orange in color. This color change and UV-Visible spectrum isillustrated in FIG. 14. The shift in color was readily visible to thenaked eye, and thus may be easily detected without the use of equipmentsuch as spectrometer.

Ultraviolet-visible (UV-Vis) spectroscopy was utilized to quantify thefilms' exposure to a range of aqueous concentrations of glucose (0-50mg/ml). It was predicted that the spectrum peak should red-shift afterexposure to glucose due to the binding and incorporation of the glucosemolecule causing swelling. When exposed to concentrations of 50 mg/mland 40 mg/ml glucose, the films shifted from green to orange or yellow,respectively, as shown in FIG. 15. This indicates that the films becameswollen as expected. However, exposure to lower concentrations ofglucose had a different effect.

As shown in FIG. 15, after exposure to concentrations of 30, 10 and 5mg/ml glucose, the films blue-shifted, indicating that the polymer filmhad collapsed when exposed to these concentrations of glucose solution.Interestingly, this counterintuitive observation of swelling at highconcentrations and collapsing at lower concentrations is provided inboronic acid detection systems.

A bi-modal response in a polymerized crystalline colloidal hydrogelsystem has been demonstrated. When the hydrogel was exposed to highconcentrations of glucose, each boronic acid functionality bound to oneglucose molecule, placing a negative charge on the boron atom whichcaused the film to swell and red-shift in color. Exposure to lowerconcentrations caused two boronic acid functionalities to bind to oneglucose molecule. This effectively creates crosslinking in theirhydrogel system, causing it to collapse and blue-shift in color.

Based on the results disclosed herein and illustrated in FIG. 15, asimilar phenomenon is occurring in the functionalized block copolymersystem. The effect of pH on phenylboronic acid's ability to bind sugarssuch as glucose is demonstrated. It has been shown that higher pHincreases the K_(eq) of the glucose binding reaction, while lower pHdecreases the K_(eq) of binding glucose.

Selectivity

The phenylboronic acid functionality introduced to the P2VP block of theBCP can covalently bind to any diol-containing sugar (e.g., such asglucose, fructose, mannose and galactose). The K_(eq) of boronic acidbinding to each one of these sugars varies depending on the sugar. Thissuggests that the boronic acid functionalized film should have aselective response to each simple sugar. To observe this effect, a BCPfilm functionalized and cross-linked to yield a blue color in water wasexposed to 50 mg/ml solution of glucose, fructose, mannose, andgalactose. After 30 minutes of exposure, the BCP film's visible spectrawas measured using UV-Vis spectroscopy. The peak difference inwavelength was then calculated as the difference between the peakwavelength of the film in water and in the sugar solution.

As shown in FIG. 16, the BCP film exposed to fructose exhibited thelargest response of a 200 nm red-shift in wavelength. The film exposedto galactose exhibited a 70 nm red-shift in color. The smallestresponses were those films exposed to glucose and mannose, whichexhibited 44 nm and 37 nm decreases, respectively. In additionalexperiments, the 50 mg/ml glucose solution evoked the same shift inwavelength (see FIGS. 15A and 16), although in each case the originalcolor of the BCP film in water was different.

This difference in response between fructose, galactose and glucose wasexpected. It has been reported that fructose has the highest K_(eq) tobind to boronic acid, followed by galactose, and then glucose (seeSpringsteen, G., Wang, B. H. (2002) Tetrahedron 58(26):5291-5300). Thedifference in binding is due to the steric structure of each sugar.Boronic acid binds to both 1,2 and 1,3 cis diols, but preferentiallybinds to 1,2 cis diols. Fructose has a planar 1,2 cis diol. Glucose doesnot have a 1,2 cis diol, but has a 1,3 cis diol in its dominate pyranoseform. Generally, glucose binds to boronic acid in its furanose form,which does contain a planar 1,2 cis diol (see Bielecki et al. (1999)Journal of the Chemical Society: Perkin Transactions 2(3):449-455). Thisresult signifies that for a given concentration, the boronic acidfunctionalized BCP film can differentiate between glucose, galactose andfructose.

The binding and color change indicate that PS-b-2VP block copolymerfilms may be utilized for detecting a wide variety of chemical orbiological targets or threats. The films functionalized with the2-bromomethylphenylboronic acid demonstrated the ability to respond toglucose with a change in color without the use of any supplementaryenzymes such as glucose oxidase. Functionalization with bromoethylaminewas further modified with polyclonal ovalbumin antibodies. This filmresponded to the ovalbumin protein with a change in color. This conceptcan be further adapted for the detection of auto-inducer2 (AI-2),glycerol, Ricin or TATP.

The phenylboronic acid functionalized films can be modified to detectAI-2, which includes molecules having chemical structures isomeric tosimple sugars and described as a universal signaling molecule in manytypes of foodborne pathogens, giving this film potential application infood packaging. This functionalization can also be used for detection ofglycerol, a precursor to nitroglycerin for application in explosivesdetection. The protein attachment protocol can also be further modifiedfor the attachment of peroxidase class enzymes. These enzymes can breakdown TATP, producing alcohols which can swell and change the color ofthe polymer films.

The disclosed colorimetric material system is unique in that it requiresonly the functionalized block copolymer to indicate an exposure to achemical or biological target. For example, once functionalized with the2-(bromomethyl)phenylboronic acid, the PS-b-P2VP film demonstrates theability to respond to glucose with a change in color without the use ofany supplementary enzymes such as glucose oxidase or additionalequipment to assess the visible color change. The block copolymerlamellar stacks responded with a red-shift in color in highconcentrations of glucose and a blue-shift in color for lowconcentrations of glucose. The BCP film also exhibited a selectiveresponse to fructose, glucose or galactose, by swelling to differentdegrees depending on which sugar is present.

The PS-b-P2VP block copolymer can easily be processed into films, sheetsor other large area coatings as needed. The color of the BCP film can betuned to blue, green, yellow or orange as demonstrated. While similarsensors exist in porous silicon, lithography and hydrogel systems, theylack the ease of fabrication and use of these block copolymer films andcan require the use of a spectrometer to measure the shift in color. Theresults show the capabilities of photonic BCP films for chemical sensingas the functionalization can be exchanged for different sensingmoieties.

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference in its entirety. While theinvention has been described in connection with specific embodimentsthereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

What is claimed is:
 1. A method of providing a sensor system fordetecting a target analyte, comprising the steps of: providing a blockcopolymer, the block copolymer having a lamellar morphology including atleast two layers; functionalizing the block copolymer; and linking anantibody to the functionalized block copolymer, wherein the antibody iscapable of binding a moiety of a target analyte, and wherein thedistance between the layers changes upon exposure to and selectiverecognition of the target analyte by the antibody so that thefunctionalized block copolymer exhibits a shift in peak wavelength inthe visible spectrum.
 2. The method of claim 1, wherein the blockcopolymer is 2-vinyl pyridine.
 3. The method of claim 2, wherein the2-vinyl pyridine block copolymer is functionalized with2-bromomethylphenylboronic acid or bromoethylamine.
 4. The method ofclaim 1, wherein the target analyte is selected from the groupconsisting of glucose, fructose, galactose and mannose.
 5. The method ofclaim 1, wherein the target analyte is selected from the groupconsisting of a foodborne pathogen, a toxin, and an explosive compound.6. The method of claim 5, wherein the foodborne pathogen is selectedfrom the group consisting of Escherichia coli, Listeria, and Salmonella.7. The method of claim 5, wherein the toxin is selected from the groupconsisting of ricin, sarin, and soman.
 8. The method of claim 5, whereinthe explosive compound is selected from the group consisting ofnitroglycerin and triacetone triperoxide.
 9. The method of claim 1,comprising the further step of coupling the functionalized blockcopolymer to a textile material.